Methods and Products for Controlling Silk Fly and Phorid Fly in Maize

ABSTRACT

The invention relates to methods for producing maize plants, plant materials and seeds that provide a means of controlling and defending against the corn silk fly and phorid fly, and improved maize plants, plant materials and seed produced by these methods. Corn silk flies and phorid flies are highly detrimental and destructive insects damaging maize plants, plant materials and seed during their growing stages in many different geographic regions worldwide. The inventive methods utilize molecular markers to incorporate genes that prove a means for controlling and defending against the corn silk fly and phorid fly into male and female inbred parent maize lines used to produce maize hybrids, or into at least one of the maize parent lines of a hybrid.

SEQUENCE LISTING

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FIELD OF THE INVENTION

The present invention is directed to novel methods for producing maizeplants, plant materials and seeds that provide a means of controllingand defending against the corn silk fly and phorid fly and improvedmaize plants, plant materials and seed produced by these methods. Theunique methods utilize molecular markers to incorporate genes to providea means of controlling and defending against the corn silk fly and thephorid fly into the male and/or female parent lines of maize hybridplants, plant parts and seeds, or into at least one of the maize parentlines of a hybrid. The unique methods also provide a means for testingand identifying the presence of the genes which are responsible forcontrolling and defending against the corn silk fly and phorid fly inmaize plants.

BACKGROUND OF THE INVENTION

Maize Crops and Insect Pests

Corn (maize) is one of the world's oldest and most widely grown crops.Human beings have cultivated and used it for centuries. Maize plantshave ten chromosomes, and the maize genome was originally very diverse,consisting of a number of different types of corn, which are generallyclassified by characteristics of their kernel endosperm. The most commontypes of corn include flint (Zea mays var. indurate), flour (Zea maysvar. amylacea), dent (Zea mays var. indentata), pop (Zea mays var.everta), sweet (Zea mays convar. saccharata var. rugosa), waxy (Zea maysvar. ceratina) and pod (Zea mays var tunicata). The physical appearanceof the plants of each type varies, as do their kernels and othercharacteristics.

Over time, as maize became increasingly domesticated by human beings,much of its original genetic diversity was either lost or becameincreasingly difficult to access by plant breeders. (“GeneticVulnerability of Major Crops,” Natl. Acad. Sci., Washington, DC, ISBN0-309-02030-1, Library of Congress Catalog Card Number 72-77533, 100-01(1972)). Moreover, maize selection processes based upon genotype and/orphenotype, and structured plant breeding programs developed by researchinstitutes, universities, and private and public companies, havemodified the original diverse maize genome, resulting in diminishedmaize crop diversity and, thus, more genetically restricted maizespecies diversity. This reduction in maize crop diversity has resultedin a number of maize plant species now being grown in large homogeneousstands, which has resulted in an increased likelihood of insect pestinfestations to the maize crops. Insect pests are responsible forsubstantial maize crop losses worldwide. Insect infestations adverselyaffecting maize crops have, in the past, been addressed by breeders andgrowers largely via applications thereon of chemical pesticides and/orinsecticides manufactured and/or distributed by a number of differentcompanies. Insect infestation in maize crops has also been addressedusing molecularly engineered or transgenic products commonly referred toas “GMOs” (Genetically Modified Organisms).

It has been well documented that significant economic losses have beensuffered due to insect pests that can markedly reduce the amounts ofagricultural produce, such as maize crops. These economic losses haveadversely impacted all different types of maize crops, but areespecially acute in maize crops such as sweet corn, a substantialportion of which is typically eaten by human beings as a vegetable.

Sweet corn is a type of maize plant that is classified as Zea maysconvar. saccharata var. rugosa, and has white, yellow, bi-colored ormulti-colored kernels that typically have a high sugar content. Sweetcorn is typically eaten by human beings as a vegetable, either directlyfrom the maize cob, or by having the sweet kernels removed from the cob,and may also be either canned or frozen, and is a major vegetable cropthat is grown all over the world primarily for fresh consumption. Thefruit of the sweet corn plant is the corn kernel, and the ear consistsof a collection of kernels on the cob. The ear is covered by tightlywrapped leaves (the husk).

Because sweet corn is most often eaten fresh on the cob, the physicalappearance of its kernels, its cob and its husk surrounding the cob isvery important to consumers worldwide. Husks, kernels and cobs that showinsect infestation and/or damage are typically not desired by consumers,and are often rejected by retail purchasers and consumers at the pointof sale (i.e., plant stand, supermarket, grocery store, or the like),thereby leading to substantial wasted product, spoilage and resultingeconomic losses. To be desirable to consumers, maize husks need toappear unblemished, with few or no holes or other damage, caused byinsects. The kernels on the maize ears must generally also be smooth,plump and unwrinkled to be desired and purchased by retail buyers andconsumers. Evidence of insect damage to maize husks, kernels and/or cobsoften leads to unmarketable product and resulting economic loss.

Corn Silk Flies

Corn silk flies are destructive, metallic green to black colored flieshaving distinctive wing patterns, and wing flapping behavior and apreference for sweet corn ears. They are increasingly becoming a majorinsect pest in significant sweet corn production areas worldwide, andsweet corn consumers have virtually no tolerance for corn silk flydamage. In the past 50 years, the sweet corn industry has growndramatically worldwide and, thus, the corn silk fly has becomeincreasingly more important. Numerous species of this insect pest existprimarily in tropical and subtropical areas of the western hemisphere.Corn silk flies can be found throughout Florida, Puerto Rica, theCaribbean Islands, Mexico and Central and South America to Argentina andChile, and are also prevalent in other parts of the world. Sweet cornand other maize types are the most susceptible to damage by the cornsilk fly during the first few days after its silk emergence, and priorto silk senescence. Entire fields of sweet corn can be destroyed, andnot considered for harvest by breeders and/or growers, if they gountreated for these destructive pests, or if individual treatments aremissed due to weather, time or other constraints. Similarly, maize earsproduced for seed and silage are also vulnerable to attack by theseinsects, which can result in significant yield reductions or withproduct being destroyed. A present continuous and growing market demandfor fresh sweet corn has required a use of sequential planting of sweetcorn crops that moves northward from the southern tip of Florida intoGeorgia, thereby facilitating the movement, and spread, of the corn silkfly into new and more temperate production regions, aiding its adaptionto cooler weather patterns, and increasing a need for specific insectcontrol strategies and host plant resistance. (B. T. Scully et al.,“Resistance in Maize to Euxesta stigmatias Loew (Diptera: Otitidae),” J.Entomol. Sci. 35(4): 432-443 (2000)).

Numerous species of corn silk flies are prevalent in Florida, includingChaetopsis massyla, Euxesta annonae, Euxesta eluta, Euxesta stigmatiasand others. The genus Euxesta is represented by 36 corn silk fly speciesin North America (north of Mexico), and 69 such species in the Americassouth of the United States (central and South America). The genusChaetopsis is represented by seven corn silk fly species in NorthAmerica, and ten such species in the Americas south of the UnitedStates, with four such species common to both. An even more damagingcorn silk fly known as the phorid silk fly has been identified insouthern Florida.

The corn silk fly can inflict immense damage on sweet corn. Such damagecan result in “poor kernel set,” thereby undesirably producingasymmetric kernel size and/or rows on the cob.

Further, adult females typically lay clusters (generally 8 to 40) oftheir white oblong eggs, generally about 1.0 mm long, on corn silkemerging from sweet corn husks, and underneath the husks protecting thesilk channel. (B. T. Scully et al., “A Rating Scale to Assess DamageCaused by the ‘Corn Silk Fly’ (Euxesta stigmatias Loew) (Diptera:Otitidae) on the Ears of Sweet Corn,” Subtropical Plant Sci. 54:34-38(2002)). Corn silk fly larvae subsequently hatch (usually in two to fourdays) and resulting ear-feeding maggots typically begin feeding on, anddown, the corn silk, ultimately penetrating the corn kernel pericarp andfeeding on developing kernel endosperm. Up to several hundred maggotscan feed on a single sweet corn (or other maize) ear, and the larvalstage commonly lasts about 20 days, after which the larvae typicallyexit the sweet corn ears, jump off of the plants, and pupate the soil(Scully et al. 2002). Adults generally emerge in about five to ninedays, and the life cycle of the corn silk fly is usually completed inless than three weeks, with reproductive adults possibly living up tofour weeks (Scully et al. 2000). Large numbers of corn silk fly larvaegenerally result in wet, decomposing corn silk within the silk channelof the corn ears, thereby making such ears unmarketable without firsttrimming the ears to remove their tips. Such trimming undesirablyresults in a poor ear appearance and diminishes ear size. Corn silk flylarvae also feed on the tips of the cobs, resulting in “blank tips.”Larvae that feed down into the ears typically cut into the pericarps ofdeveloping corn kernels, and often hollow out their interiors(endosperm), thereby destroying them. Corn silk larvae may also bedisadvantageously found feeding along the entire length of corn ears,and resulting extreme damage thereto can very undesirably result intwisted, unsightly ears with few, if any, kernels.

Phorid Flies

Phorid flies (Diptera) are similarly very destructive insects thatdamage the silk tissue, the tips of the cob, and have been observed todisrupt pollination, leaving the top of the cob 25-50% bare of kernelsat harvest. (David Owens et al., “Cob Flies, Megaselia spp. (Diptera:Phoridae), in Sweet Corn,” Univ of Fla Entomol & Nematol 5 (2016).) Likethe corn silk fly, the phorid fly is increasingly becoming a majorinsect pest in significant sweet corn production areas worldwide andsweet corn consumers have virtually no tolerance for phorid fly damage.Phorid flies have a very distinct appearance and behavior. Adult phoridflies are 2-3 mm in length, about half the length of silk flies (4-7mm). (Owens et al. 2016 at 3.) Adult phorid flies are light brown withdark brown to black markings on the dorsal side of the abdomen. Theirhind femurs have a dark pigmentation at the end. They have no dark bandsacross their wings, further distinguishing themselves from silk flies.The wings of phorid flies have heavy veins that curve to meet the wingmargin about half way between the wing tip and the body. The head of thephorid fly is small and has large, black bristles. Phorid flies have avery distinctive locomotion. They move very quickly in a “herky-jerky”fashion characterized by short, rapid running, a pause, a turn, and moreshort, rapid running. For this reason, they are known as humpback fliesor scuttle flies. (Owens et al. 2016 at 1.) Phorid flies are alsosometimes called “cob flies” because of the damage that they inflict onthe cobs of maize plants.

Phorid flies are part of the genus Megaselia, which is very diverse,with more than 1400 described species. (Owens et al. 2016 at 1.) Thespecies (Megaselia seticauda Malloch) has been observed to damage maize.Megaselia seticauda was first observed in Costa Rica in 1914. (Owens etal. 2016 at 1.) There were later reports of damage by phorid flies toimmature “green corn” ears in Texas (1944), Mexico (1942) and Ecuador(1954). Phorid flies were observed in Brazil (1962) and detected inCalifornia (1996). (Owens et al. 2016 at 1.) By 1950, in Texas, fieldsthat were not treated with DDT for corn earworm were severely infestedwith both M. seticauda and the silk fly Euxesta stigmatias (Diptera:Ulidiidae). Larvae of both E. stigmatias and M. seticauda were reportedas being relatively unaffected by DDT spray residues, leading to therecommendation to treat adults. (Owens et al. 2016 at 1.)

Phorid flies have increasingly become a major insect pest in Floridawhere crops are being grown for organic and other specialty markets,without the protection of insecticides. Phorid flies have been found inboth sweet corn and field corn in Florida where insecticides have notbeen applied. (Owens et al. 2016 at 1.) In April 2016, ears ofearly-silking sweet corn in a variety trial where an organophosphateinsecticide had been applied at the UF/IFAS Everglades Research andEducation Center in Belle Glade, Fla., were found to be heavily damagedby phorid larvae. (Owens et al. 2016 at 1.) The phorid larvae wereobserved to develop faster and cause damage quicker than silk fly.

Phorid fly larvae are distinguishable from larvae of silk flies by thetapering shape of the posterior abdomen. (Owen et al. 2016 at 2.) Incontrast, silk fly larval abdomens terminate bluntly, and twodark-colored, peg-like spiracular (breathing tube opening) plates can beeasily seen at the end of the abdomen. Phorid flies retain their whitecoloration throughout the larval stage. When the larvae emerge from theeggs, they enter the ear to feed on silks, cob and kernels. Once theyreach the ear, larvae feed on the cob and developing kernels at the tip.Phorid flies leave the ear 10-14 days after first silk, usually beforeblister stage, by leaving a visible shiny mucous trail on the dryingsilks. Damaged silk tissue is reddish brown to brown. (Owens et al. 2016at 4.) Heavily damaged silk appears wet and slimy. Severe tip damage canoccur on ears that are still at least one week to ten days from harvest.Because larvae develop faster and leave the ear earlier than silk fly,phorid flies may be partly responsible for the damage often attributedto silk fly.

Growers experiencing insect pressure from the phorid fly havetraditionally used insecticides as a means of control. Insecticides suchas DDT are no longer acceptable to be applied to crops in many growingregions around the world. Other less toxic insecticides sometimesrequire multiple applications to control these insects.

Agricultural Pesticides and Insecticides

In the 20th century, benefits for agricultural crops appeared to beproduced using agricultural chemical pesticides and/or insecticides tocontrol agricultural insect pests. A large crop protection industrydeveloped around chemical pesticides and insecticides during the mid tolate 20th century. Increasingly, however, and disadvantageously,chemical pesticides and insecticides came to be viewed by scientists asa potential threat to both living organisms and the environment.Pesticides and insecticides are presently heavily tested, and regulatedby governmental regulatory agencies before they can be registered forapplication. In several instances, previously-approved pesticides and/orinsecticides have been reported by researchers to pose a potential riskto the health of humans, animals and other forms of life, including apotential risk of cancer.

Most clinicians and researchers hold the opinion that no segment of thepopulation is completely protected against exposure to pesticide and/orinsecticide use, and the potentially serious detrimental health effectsthereof. (See W. Aktar et al., “Impact of pesticides use in agriculture:their benefits and hazards,” Interdiscip Toxicol. 2(1):1-12 (2009).)Higher risk population groups include agricultural farm workers,production workers, formulators, sprayers, and loaders. Certainpesticide and/or insecticide compounds and residues, known as“organochlorine compounds,” have a potential to pollute the tissues oflife forms on land (human beings and animals), in the air (birds, batsand the like) and in lakes and the oceans (fish, seals, whales, othersea mammals and other marine life forms). Certain other pesticide and/orinsecticide compounds, known as “endocrine disruptors,” have beenreported by researchers to disadvantageously elicit adverse effects bymimicking and/or antagonizing natural hormones that are present in thehuman body. Research suggests that long-term, low-dose exposure to suchpesticides and/or insecticides very disadvantageously can be associatedwith adverse health effects to human beings and animals, such as immunesystem suppression, hormone disruption, mental disorders, reproductiveabnormalities and/or cancer. In food commodities, pesticide andinsecticide use has had a serious and substantial impact. In addition,the use of specific pesticides and insecticide has been reported toresult in contamination of soil, water, turf and other forms ofvegetation.

Applying pesticides and insecticides to growing crops is expensive, andoften must be repeated, sometimes weekly, to achieve effective controlof the targeted insect. Due to physical and time constraints that areoften associated with treating large areas of maize plants with groundmachinery, such chemicals are sometimes applied aerially, which is alsoexpensive, potentially wasteful of the chemicals, and potentiallydangerous for the pilots (Scully et al. 2000).

Genetic Engineering of Insect Resistance Traits into Agricultural Plants

In the 20th century, due in part to adverse health concerns associatedwith chemical pesticides and insecticides, agricultural plants,including maize, had been modified using genetic engineering technologyto incorporate a new trait of insect resistance into the agriculturalplants themselves.

To accomplish the above, one or more genes from outside of the maize (orother plant) genome were artificially introduced into maizetransgenically using a set of several biotechnology techniques that arecollectively referred to as “recombinant DNA technology” techniques. DNAspliced to the coding portion of such genes that served to regulate howthey functioned were also transferred into the new host plant. Theinserted genes, called “transgenes” when they were inserted into the newhost plant, may have come from another plant of the same or a differentspecies, or a completely unrelated kind of organism, such as bacteria oran animal. The gene being transferred into the new host may have had itsgenetic code altered to modify its function, in addition to havingdifferent regulatory sequences spliced thereon to control how it wasexpressed (switched “on” or “off”) in the new host plant.

The process of moving genes from one species to another is called“transformation.” Once a “transgenic plant” is created, the “transgenes”can be inherited along with the rest of the plant's genes through matingby pollination. The host plant's offspring are also “transgenic” whenthey acquire the “transgenes” in this manner. As a result of theforegoing, plant breeders could take a “transgenic plant” made in thelaboratory and use various breeding techniques to develop differenttransgenic varieties of the plant that are adapted for specific uses,all with the new plant trait provided by the introduced genes fromoutside of the plant.

Plants commercialized with the above technology have typically beenreferred to as GMOs. GMOs are essentially any organism that hasundergone a recombinant DNA procedure. Recombinant DNA technologyinvolves the transfer of genetic material from one organism to anotherplant or animal. The genetic engineering process can be achieved byutilizing viruses and/or bacterial DNA to implant the desired gene(s)into the target host plant or animal. Genetic engineering has beenperformed in plants for food crops, trees, grasses, flowers, industrialproducts, pharmaceuticals, and environmental remediation andconservation.

In general, through the use of recombinant DNA technology, agriculturalresearchers have reported some improvements in forestry and tree-relatedcharacteristics associated with insect pest and disease (virus, bacteriaand fungi) resistance. Additionally, such researchers have correlatedthe use of recombinant DNA technology with some increased energyproduction, increased efficiency of pulp milling, straighter trees forlumber and building and transgenic modifications of tree fruits toimprove flavor and color. Genetic engineering in grasses and flowers hasreportedly achieved improvements in: herbicide, insect pest, and diseaseresistance, stress tolerance (enhanced tolerance to heat, cold, anddrought), and product characteristics.

In relation to industrial products, genetic engineering is beingconducted to produce proteins, biopolymers, plastics, fatty acids, oils,waxes and dyes.

There has been a substantial amount of criticism by consumers regardingthe use of genetically modified products in food, however, and manyconsumers globally are not in favor of GMOs. Critics of geneticallymodified foods advocate that the risks have not yet been adequatelyidentified and/or managed. Questions have also been raised by consumersas to the objectivity and effectiveness of food product (and other)regulatory authorities. Some organizations advocate that there are toomany unanswered questions regarding the potential adverse long-termeffects on health due to the ingestion of genetically modified foods,and have proposed mandatory labeling, or even a moratorium on such foodproducts.

Need for Alternative Insect Control and Defense Methods for Maize Crops

The use of chemical pesticides and insecticides, and of GMOs, to protectmaize crops from insect pest damage are established practices. However,consumers and retail buyers have expressed serious health andenvironmental concerns over these traditional means of insect pestcontrol.

There has, therefore, been a long-felt and unfulfilled need for a morewholesome and health-conscious approach to insect pest control, and tothe production of food products that do not contain either pesticide orinsecticide residue on the one hand, or genes that have beentransgenically introduced into the maize genome from sources outside ofthe maize genome on the other hand. With respect to sweet corn, inparticular, and maize generally, there has been a long-felt need in thesweet corn and maize industries for a wholesome and health-consciousmethod for providing a means for controlling and defending againstinsect pests, such as the corn silk fly and the phorid fly (all speciesand types).

Plant breeders have attempted for many years to develop a maize geneticsource of insect pest resistance. In 1992, N. W. Widstrom et al.,“Recurrent Selection for Resistance to Leaf Feeding by Fall Armyworm onMaize,” Crop Sci. 32:1171-1174 (1992), reported indications that the useof “recurrent selection breeding” methodologies (reselection generationafter generation, with intermating of selected plants to produce thepopulation for the next cycle of selection) could be useful indeveloping insect protection utilizing an “exotic maize synthetic”(uncommercialized wild-type accessions collected from around the world).However, while Widstrom expressed hope that “recurrent selectionbreeding” would accomplish the foregoing goal, time passed, and thereare no significant successes reported of increasing control over ordefending against insect pests utilizing such conventional plantbreeding methodologies.

Because no commercially-available sweet corn cultivars were known toprovide control over or defense against the corn silk fly, controlpractices focused instead on insecticide use, rather than on host plantresistance (Scully et al. 2000; Scully et al. 2002). Very few studiescited maize lines having any inherent resistance at all to the corn silkfly. A study in Brazil reported that two sweet corn test hybrids showedsome resistance to an unknown species of Euxesta, however, no sweet cornvarieties having control over or the ability to defend against Euxestahave ever been commercialized in the United States or any other parts ofthe world. (See M. C. et al., “Avaliacao da Resistencia a Helicoverpazea (Boddie) Lepidoptera: Noctuidea) e Euxesta sp. (Diptera: Otitidae)em linhagens de milho-doce,” An. Soc. Entomol. Brasil 23(1):136-140(1994).) No host plant resistance to E. stigmatias has ever beenidentified in sweet corn or maize generally (Scully et al. 2000).

In 2000, agricultural researchers considered whether field corn couldprovide a source of resistance to corn silk fly for improvement of sweetcorn in regard to damage from corn silk flies (Scully et al. 2000). Thestudy concluded that, in order to lower the susceptibility of sweet cornto the corn silk fly, many different factors still needed to be betterunderstood, for example, the role of the endosperm mutant genes, andtheir pleiotropic effect, on silk biochemistry, and the genetic basisof, and mechanism of resistance to, the corn silk fly in the corn ear.Researchers thereafter continued their efforts to find a source of cornsilk fly resistance from the maize genome to effectively control suchinsect pest.

To date, efforts to achieve corn silk fly protection in maize crops to acommercially-acceptable level have been limited by a number of differentfactors. Hindrances to maize breeders and growers have included no, orlimited, access to insect rearing facilities, negative characteristicsassociated with donor genetic and other materials, complications frommultiple gene factors and gene interactions, epistasis (a certainrelationship between genes in which an allele of one gene hides or masksthe visible output, or phenotype, of another gene), environmentalinfluences and considerable time requirements. In addition, furthercomplications exist, such as incomplete knowledge and characterizationof the actual insect pest protection gene sequences and their specificlocations on the respective chromosomes.

Control and Defense of Other Types of Insect Pests in Maize

Agricultural researchers have conducted studies to try to control othertypes of insect pests, such as Lepidopteran larvae, in maize crops.(Lepidoptera is an order of insects that includes moths and butterflies,both called lepidopterans.) With respect to fall armyworm, Spodopterfrugiperda (J. E. Smith) (Lepidoptera: Noctuidae), and southwestern cornborer, Diatraea grandiosella Dyar (Lepidoptera: Crambidae), QTL regionson chromosomes 1, 5, 7, and 9 in maize inbred line Mp708 and itsresistant parent Mp704 have been identified as conferring resistance toboth insects. (T. Brooks et al., “Genetic Basis of Resistance to FallArmyworm (Lepidoptera: Noctuidae) and Southwestern Corn Borer(Lepidoptera: Crambidae) Leaf-Feeding Damage in Maize,” J. Econ.Entomol. 100(4):1470-1475 (2007)). However, no studies have reported anycorrelation between resistance to fall armyworm and southwestern cornborer and corn silk fly and phorid fly resistance.

Furthermore, researchers have identified a 33 kDa cysteine protease (anenzyme that degrades proteins) called “maize insect resistance cysteineprotease” (Mir1-CP) as having plant protection attributes. Results ofthese studies indicated that, in specific maize lines, there is a ratherrapid accumulation of Mir1-CP in the whorls of their husk leaves inresponse to feeding by Lepidopteran larvae. Researchers observed thatthis naturally-occurring cysteine protease is located in the maize rootsand vascular tissues, and is readily mobilized in response to insectherbivory. In vitro studies suggested that Mir1-CP completely permeatesthe insects' “peritrophic matrix” (PM), an extracellular envelope thatlines the digestive tract of most insects, and is composed largely ofproteins and glycosaminoglycans embedded in a chitinous matrix, andprotects the insects' midgut epithelium from mechanical damage,pathogens and toxins. These studies also suggested that Mir1-CP plays anactive role in maize plant digestion and nutrient absorption bydigesting (breaking down) PM proteins to retard insect growth. Studiesalso indicated that Mir1-CP was most effective on Lepidopteran larvaebelonging to the Noctuidae family, the largest and most economicallyimportant family of Lepidopterans.

Mir1-CP is a papain-like cysteine protease that has amino acid sequencesin common with several other baculoviruses that infect Lepidopteranlarvae. However, Mir1-CP is the only reported defensive cysteineprotease that has shown to directly damage the Lepidopteran PM. U.S.Pat. No. 5,977,440, issued in 1999, describes the cDNA encoding Mir1-CP,and a method of conferring insect resistance on a plant susceptible toLepidopteran feeding by expression of Mir1-CP in the plant.

Since Mir1-CP was identified, few maize parent lines or hybrids wereever commercialized using the Mir1-CP gene, and no sweet corn parentlines or hybrids containing Mir1-CP were ever commercialized. Instead,the field corn and sweet corn industries continued to use chemicalpesticides and insecticides and GMOs as the basic source of insectprotection in maize.

Despite the efforts of many agricultural researchers, prior to thepresent invention, no form of commercially acceptable genetic controlhas been identified as a means for controlling infestations of the cornsilk fly or the phorid fly in maize crops. The present invention,therefore, provides a long felt, but unresolved, need in the maizebreeding and growing industries for a more wholesome and healthconscious approach to insect pest control in maize plants, and to theproduction of maize ears and kernels (and other food products) that donot contain pesticide and/or insecticide residue or genes that have beentransgenically introduced therein from sources outside of the maizegenome.

SUMMARY OF THE INVENTION

The present invention provides unique, cost-effective, reliable andsuccessful methods for breeding, growing, developing and producing maizeplants, plant materials and seeds that very advantageously provide awholesome, natural and health conscious means of controlling anddefending against corn silk flies and phorid flies, and to the improvedmaize plants, plant materials and seeds that are produced by thesemethods, which have one or multiple desirable characteristics forconsumers of these products, as well as for commercial plant growers andhome gardeners. The inventive methods do not employ pesticides and/orinsecticides, or genes that have been transgenically introduced intomaize plants, plant materials or seeds from sources outside of suchplants, plant materials or seeds, to control corn silk flies and phoridflies, and the resulting products, thus, do not contain pesticide orinsecticide residues, and are not GMOs. In contrast, the inventivemethods utilize one or a plurality of molecular markers to assist inincorporating genes to provide a means of controlling and defendingagainst the corn silk fly and phorid fly into the male and/or femalemaize parent lines of maize hybrid plants, plant materials or seeds(i.e., into at least one of the two maize parent lines of such hybrids).The unique methods also provide a means for testing and identifying thepresence of the genes which are responsible for controlling anddefending against the corn silk fly and phorid fly in maize plants.

The present invention provides a novel construction of genetic elementsthat confers a high, or an enhanced, degree of maize plant protectionfrom the corn silk fly (all species and types) onto maize plants, plantmaterials and seeds, thereby preventing, or reducing, significant orsubstantial losses in yield and quality of maize plant crops, and theirrelated plant materials and seeds, and a corresponding loss in revenuesfrom sales of maize plants, ears and seed.

In one aspect, the present invention provides a method for producing ahybrid maize plant, plant material or seed having a maize genome-basedform of controlling and defending against corn silk flies and phoridflies without the use of chemical pesticides or insecticides.

In another aspect, the present invention provides a hybrid maize plant,plant material or seed that provides control over and defends againstcorn silk flies and phorid flies in comparison with a conventionalhybrid plant, plant material or seed.

In still another aspect, the present invention identifies singlenucleotide polymorphisms (SNPs) and indels that may be used in a markerassisted selection process to produce inbred maize parent lines, hybridmaize plants, plant material or seed that provides control over anddefends against corn silk flies and phorid flies.

In another aspect, the present invention provides hybrids and inbredlines of maize plants, plant material or seed comprising quantitativetrait locus regions that control or defend against corn silk fly andphorid fly.

In another aspect, the present invention provides a method for producinga hybrid maize plant, plant material or seed comprising quantitativetrait locus regions that control or defend against corn silk fly andphorid fly, comprising the following steps:

-   -   (a) identifying one or more maize parent lines containing one or        more of the SNPs or indels that have been identified as        providing control or defense against corn silk fly and phorid        fly utilizing molecular markers;    -   (b) incorporating one or more of the SNPs or indels that provide        control or defense against corn silk fly and phorid fly into a        male or female inbred maize parent line;    -   (c) crossing the male or female inbred maize parent line        containing one or more of the SNPs or indels that provide        control or defense against corn silk fly and phorid fly with        another inbred maize parent line to produce a hybrid; and    -   (d) optionally, conducting one or more genetic identification        tests to confirm that the hybrid contains the SNPs or indels        that provide control or defense against corn silk fly and phorid        fly.

In another aspect, the present invention provides a method for producinga male or female inbred maize parent line that provides control ordefense against corn silk fly and phorid fly, comprising the followingsteps:

-   -   (a) identifying one or more maize lines containing one or more        of the SNPs or indels that have been identified as providing        control or defense against corn silk fly and phorid fly        utilizing molecular markers;    -   (b) incorporating one or more of the SNPs or indels that provide        control or defense against corn silk fly and phorid fly into a        male or female inbred maize parent line;    -   (c) utilizing recurrent selection or other plant breeding        methods to produce a male or female inbred maize parent line        containing one or more of the SNPs or indels that provide        control or defense against corn silk fly and phorid fly; and    -   (d) optionally, conducting one or more genetic identification        tests to confirm that the inbred maize parent line contains the        SNPs or indels that provides control or defense against corn        silk fly and phorid fly.

In another aspect, the present invention provides a hybrid maize plant,plant material or seed prepared by a process comprising the steps of:

-   -   (a) identifying one or more maize parent lines containing one or        more of the SNPs or indels that have been identified as        providing control or defense against corn silk fly and phorid        fly utilizing molecular markers;    -   (b) incorporating one or more of the SNPs or indels that provide        control or defense against corn silk fly and phorid fly into a        male or female inbred maize parent line;    -   (c) crossing the male or female inbred maize parent line        containing one or more of the SNPs or indels that provide        control or defense against corn silk fly and phorid fly with        another inbred maize parent line to produce a hybrid; and    -   (d) optionally, conducting one or more genetic identification        tests to confirm that the hybrid contains the SNPs or indels        that provide control or defense against corn silk fly and phorid        fly.

In another aspect, the present invention provides a male or femaleinbred maize parent line prepared by a process comprising the steps of:

-   -   (a) identifying one or more maize lines containing one or more        of the SNPs or indels that have been identified as providing        control or defense against corn silk fly and phorid fly        utilizing molecular markers;    -   (b) incorporating one or more of the SNPs or indels that provide        control or defense against corn silk fly and phorid fly into a        male or female inbred maize parent line;    -   (c) utilizing recurrent selection or other plant breeding        methods to produce a male or female inbred maize parent line        containing one or more of the SNPs or indels that provide        control or defense against corn silk fly and phorid fly; and    -   (d) optionally, conducting one or more genetic identification        tests to confirm that the inbred maize parent line contains the        SNPs or indels that provide control or defense against corn silk        fly and phorid fly.

In another aspect, the present invention provides hybrid and inbred lineZea mays or Zea mays, convar saccharata var. rugosa maize plants thatare produced by the methods described above. Among the products of thepresent invention are Sweet Corn Hybrid NBDX 1001 and Sweet Corn HybridNBDX 1002 and inbred parent lines Sweet Corn NBD 01, Sweet Corn NBD 02and Sweet Corn NBD 03. In addition, products of the present inventioninclude hybrid and inbred maize lines that are capable of producing earsand kernels having a damage rating of 2 or less or 1 or less.

In yet another aspect, the present invention includes comprising hybridmaize plants, plant materials and seed having a genetic backgroundcomprising SNPs or indels including one or more of SEQ ID NOS. 6-12.

In yet another aspect, the present invention provides a plant, plantmaterial or seed that is produced by any one of the methods that isdescribed above.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a statistical model of a genome-wide association study (GWAS)of the association of each SNP across all ten chromosomes that arepresent in maize plants, and using phenotypic data resulting from afirst maize plant corn silk fly and phorid fly damage scoring scale(“the first scoring scale”) having values ranging from 1 to 8 (Phe1-8)based upon the level of susceptibility or lack of defense of a maizeplant variety to corn silk fly and phorid fly damage. In this scoringscale, a score of 1 represents a maize plant variety that is the mostresistant, or has the greatest defense capabilities, to corn silk fliesand phorid flies, a score of 8 represents a maize plant variety that isthe most susceptible, or the least defensive, to corn silk flies andphorid flies (i.e., that is not at all resistant to corn silk flies andphorid flies), and scores of 2-7 represent varying levels of resistance(defense) and susceptibility (lack of defense) to corn silk flies andphorid flies, with more resistance (defensiveness) and lesssusceptibility (lack of defense) indicated by the lower numbers, andwith less resistance (defensiveness) and more susceptibility indicatedby the higher numbers. This GWAS analysis was performed with the dataset from the first scoring scale.

FIG. 2 is a statistical model of a GWAS of the association of each SNPacross all ten chromosomes that are present in maize plants, and usingphenotypic data resulting from a second maize plant corn silk fly andphorid fly damage scoring scale (“the second scoring scale”) in whichthe above-described first scoring scale was recoded to create a binarydata set necessary for the statistical analysis model, which requires abinary logarithmic application, and has a score of either 0 or +1, andgrouping together in the +1 group every maize plant having a score inthe first scoring scale of greater than 1 (PheBin). This GWAS analysiswas performed with the binary data set from the second scoring scale.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention may be understood more readily by reference to thefollowing detailed description of the preferred embodiments of theinvention.

Definitions

For purposes of clarity, various terms and phrases that are usedthroughout this specification and the appended claims are defined in themanner that is set forth below. If a term or phrase that is used in thisspecification, or in the appended claims, is not defined below, orotherwise in this specification, the term or phrase should be given itsordinary meaning.

The term “allele” as is used herein refers to one of multiplealternative form of a gene (one member of a pair) that is located at aspecific position or locus on a specific chromosome, and controls thesame phenotype (with potentially differing effects). Alleles arevariants of a gene that produce different traits in a gene'scharacteristics, and can differ in either coding sequences or non-codingsequences.

The term “amplification” as is used herein means a process in molecularbiology by which a nucleic acid molecule is enzymatically copied togenerate a progeny population with the same sequence as the parentalone. The most widely used amplification method is Polymerase ChainReaction (PCR), and the result of a PCR amplification of a segment ofDNA is called an “amplicon.”

The term “backcross” as is used herein means to cross (a hybrid or otherplant) with one of its parents, or with an individual that isgenetically identical or similar to one of its parents.

The phrases “blank tip” and “blank ear tip” as are used herein mean thetip of a maize ear (one or a plurality of rows on that end of the earthat is opposite to the end that is adjacent with the stalk in whichkernels would typically be present) that has little or no physicalindication that one or a plurality of kernels were ever developed (i.e.,it is missing one or a plurality of kernels in that area of the cornear). Blank ear tips, which are missing kernels, may result fromenvironmental stress factors and/or insect pest feeding thereon, andtypically render the maize ear undesirable to maize purchasers andconsumers (the ear is not complete in regard to kernels and, thus, ithas an undesirable appearance and fewer kernels for consumption).

The term “breeding” as is used herein means the science and/or art ofmanipulating the heredity of an organism for a specific purpose.

The terms “control” or “controlling” as used herein mean the ability ofa maize plant to prevent, or assist in preventing, damage from insects,such as armyworm, corn silk fly and phorid fly.

The terms “corn” and “maize” as is used herein means any of numerouscultivated forms of a widely grown, usually tall annual cereal grass(Zea mays) bearing grains or kernels on large ears, and includes thenumerous varieties of sweet corn and supersweet corn. The grains orkernels of this plant may be used as food for humans and livestock, orfor the extraction of an edible oil or starch. The kernels of sweet cornmay be eaten raw or cooked, and may be canned, frozen and/or stored inother manners that are known by those of ordinary skill in the art.

The term “crop” as is used herein means the periodic, such as annual,bi-annual, quarterly, seasonal, or the like, yield of any plant that isgrown in significant quantities to be harvested as food, as livestockfeed, as fuel or for any other economic (or other) purpose. Many typesof crops are used for industrial purposes, for example, they are grownand harvested for the sole purpose of making profit and feeding people,and are grown in large quantities in certain areas that are suitable forgrowing crops. That which is considered to be “seasonal” in onegeographic region, such as a town, city, county, state, country orcontinent, may not be considered to be “seasonal” in a different suchgeographic region, and vice versa.

The terms “cross,” “crossing,” “interbreeding” and “crossbreeding” asare used herein mean the act of breeding different species or varietiesof plants to produce hybrids.

The phrase “damage rating scale” as is used herein refers to thedescription of the level of damage attributable to insects, includingarmyworm, corn silk fly and phorid fly, to the ear and/or kernel of amaize plant using the numerical rating scale that is set forth in Table3.

The terms “defend” or “defense” as used herein mean the ability of amaize plant to protect itself, or to be protected, from or againstdamage from insects, such as armyworm, corn silk fly and phorid fly.

The term “dominant” as is used herein means an allele or a gene that isexpressed in an organism's phenotype, generally masking the effect ofthe recessive allele or gene, when present. Usually, a dominant alleleis symbolized with a capital letter, and a recessive allele issymbolized with a small letter, for example: Hh (where H refers to thedominant allele and h refers to the recessive allele).

The phrases “DNA probe,” “gene probe” and “probe” as are used hereinmean a single-stranded DNA molecule used in laboratory experiments todetect the presence of a complimentary sequence among a mixture ofvarious single-stranded DNA molecules.

The phrase “DNA sequencing” as is used herein means a determination ofthe order of nucleotides in a specific DNA molecule.

The term “endosperm” as is used herein means the nutritive tissue thatis found in many seeds of plants, and that surrounds the embryo withinsuch seeds. It supplies nutrients to the embryo.

The term “express” as is used herein means to manifest the effects of agene, to cause to produce an effect or a phenotype, or to manifest agenetic trait, depending upon the context. The expression of a gene isthe translation of information encoded in the gene into protein orribonucleic acid (RNA).

The term “F1 hybrid” as is used herein means the first filial generationof offspring of distinctly different parental types.

The term “gene” as is used herein refers to the basic unit of heredity(genetic traits) in a living organism (plant, animal or micro-organism)that holds the information that is required to pass genetic traits tooffspring. It is a segment of deoxyribonucleic acid (DNA) thatcontributes to a phenotype/function. The DNA is a molecule in the shapeof a double helix, with each rung of the spiral ladder having two pairedbases selected from adenine (A), thymine (T), cytosine (C) or guanine(G). Certain bases always pair together (AT and GC), and differentsequences of base pairs form coded messages. Genes are arranged inprecise arrays all along the length of chromosomes, which are muchlarger structures.

The phrase “gene expression” as is used herein means the process inwhich a cell produces the protein and, thus, the characteristic, that isspecified by a gene's nucleotide sequence.

The phrase “genetic map” as is used herein means a diagram that showsthe genetic linkage relationships among loci on chromosomes (or linkagegroups) within a given species. “Mapping” is the process of defining thelinkage relationships of loci through the use of genetic markers,populations that are segregating for such markers, and standard geneticprinciples of recombination frequency. A “map location” is a specificlocus on a genetic map where an allele can be found within a givenspecies.

The phrase “genetic marker” as is used herein means a specific fragmentof DNA that can be identified within a whole genome. It is a geneticfactor that can be identified and, thus, act to determine the presenceof genes or traits linked with them, but not easily identified.

The term “genome” as is used herein means the complete set of genes inan organism, such as a plant, or the total genetic content in one set ofchromosomes, depending upon the context. It is the complete set ofchromosomes found in each cell nucleus of an individual or organism.

The phrase “genome-wide association study” or “GWAS” as is used hereinmeans an examination of genetic variation across the genomes ofdifferent individuals to identify a genetic association with aparticular phenotypic trait. A GWAS may be performed by collectingquantitative phenotypic data and genome-wide SNP data and determiningwhich SNPs have a high degree of association with the phenotypic trait.

The term “genotype” as is used herein refers to the set of genes in theDNA of an organism, plant, animal, or the like, that is responsible fora particular trait (i.e., inherited instructions that is carried withinits genetic code). A genotype is the specific combination of allelespresent at a single locus in the genome, and can only be determine bybiological testing, not by observation.

The term “germplasm” as is used herein means the living geneticresources, such as seeds or tissue, that is maintained for the purposeof animal and plant breeding, preservation, and other research uses.

The term “harvest” as is used herein means the gathering (collectingand/or assembling) of a crop of any kind, for example, of maize.

The phrase “health conscious” as is used herein means concern by anindividual about the degree of health of the individual's diet and/orlifestyles, with the term “health” referring to a state of preferablycomplete physical, mental and/or social well-being and/or an absence ofmalady, disease, defect or infirmity.

The term “heterozygous” as is used herein means having dissimilaralleles that code for the same gene or trait. It is a situation in whichthe two alleles at a specific genetic locus are not the same. An exampleis a zygote having one dominant allele and one recessive allele, i.e.,Aa, for a particular trait.

The term “heterozygosity” as is used herein means the presence ofdifferent alleles at one or more loci on homologous chromosomes.

The term “homologous” as is used herein in connection with chromosomesmeans those that contain identical linear sequences of genes, and whichpair during meiosis. It means stretches of DNA that are very similar insequence, so similar that they tend to stick together in hybridizationexperiments. Each homologue is a duplicate of one of the chromosomescontributed by one of the parents, and each pair of homologouschromosomes is normally identical in shape and size. Homologous can alsobe used to indicate related genes in separate organisms controllingsimilar phenotypes.

The phrase “homologous chromosomes” as is used herein means a pair ofchromosomes containing the same linear gene sequences, each derived fromone parent.

The term “homozygous” as is used herein means a situation in which twoalleles at a specific genetic locus are identical to one another.

The term “homozygosity” as is used herein means the presence ofidentical alleles at one or more loci (a specific place on a chromosomewhere a gene is located.) in homologous chromosomal segments.

The term “husk” as is used herein means the outer leafy protectivecovering of an ear of maize.

The term “hybrid” as is used herein means an offspring or progenyresulting from a cross between parents of two different species,sub-species, races, cultivates or breeding lines (i.e., fromcrossbreeding). A single-cross hybrid is a first generation of offspringresulting from a cross between pure bred parents. A double-cross hybridis offspring resulting from a cross between two hybrids of single cross.A three-way cross hybrid is offspring from a cross between asingle-cross hybrid and an inbred line. A triple-cross hybrid isoffspring resulting from the crossing of two different three-way crosshybrids.

The term “inbred” as is used herein means offspring produced byinbreeding (succeeding generations of organisms, such as plants, thatare all derived by breeding from the same group of closely relatedorganisms). When lines are inbred sufficiently, a homozygous conditionof particular alleles can generally be assumed.

The term “inbreeding” as is used herein means the breeding of plants,plant materials or organisms that are related, depending upon thecontext (i.e., of plants, plant materials or organisms within anisolated or a closed group of plants, plant parts or organisms). It isthe continued breeding of closely related plants, plant parts ororganisms, so as to preserve desirable traits therein.

The term “indel” as is used herein means a short polymorphism thatcorresponds to the insertion, deletion, or insertion and deletion of asmall number of nucleotides in a genomic DNA sequence.

The term “insect” as is used herein means any of a class ofinvertebrates within the arthropod phylum that have a chitinousexoskeleton, a three-part body (head, thorax and abdomen), three pairsof jointed legs, compound eyes and/or one pair of antennae, and possiblywings.

The term “insect damage” as is used herein means damage to a plant or aplant part, including the ear and silk of a maize plant, resulting frominsects including, armyworm, corn silk fly and phorid fly.

The term “introgression” as is used herein means the transfer of geneticinformation from one species to another as a result of repeatedbackcrossing of the hybrid with one of its parental species.

The term “isogenic” as is used herein means having substantially thesame genotype (i.e., genetically uniform), as all organisms produced byan inbred strain.

The terms “library,” “DNA library” and “gene library” as are used hereinrefer to a plurality or collection of DNA fragments of one or moreorganisms, each generally carried by a plasmid or virus and cloned intoan appropriate host. A DNA probe is generally used to locate a specificDNA sequence in the library. A collection representing the entire genomeof an organism is known as a genomic library, and an assortment of DNAcopies of messenger RNA produced by a cell is known as a complimentaryDNA (cDNA) library.

A “linkage map” as is used herein means a map of the relative positionsof genetic loci on a chromosome, determined on the basis of how oftenthe loci are inherited together. Distance may be measured incentimorgans (cM).

The term “line” as is used herein means a population, breed or strain ofan organism, plant or animal. A “pure line” is a population, breed orstrain of an organism, plant or animal that maintains a high degree ofconsistency in certain characters as a result of inbreeding forgenerations.

The term “locus” as is used herein refers to a specific chromosomelocation in the genome of a species where a specific type of gene can befound. It is the position on the chromosome where the gene for aparticular trait resides. A locus may be occupied by any one of severalalleles (variants) for a given gene.

The phrase “molecular marker” as is used herein means a specificfragment of DNA that can be identified within a whole genome. It is anidentifiable physical location on a chromosome (i.e., restriction enzymecutting site, gene, or the like) whose inheritance can be monitored.Molecular markers are generally found at specific locations of a genome,and are used to ‘flag’ the position of a particular gene or theinheritance of a particular characteristic. In a genetic cross, thegenes producing characteristics of interest will usually stay linkedwith the molecular markers in relatively close proximity on thechromosome. Thus, varieties can be selected in which the molecularmarker is present, since the marker indicates the presence of thedesired characteristic. Examples of molecular markers include simplesequence repeats (SSRs), SNPs, randomly amplified polymorphic DNA(RAPDs), and restriction fragment length polymorphisms (RFLPs).Additional information about the use of molecular markers for use incharacterizing and identifying maize inbred lines, validating pedigreeand showing associations among inbred lines is present in J. S. Smith etal., “An Evaluation of the Utility of SSR loci as Molecular Markers inMaize (Zea Mays L.): Comparisons with Data from RFLPS and Pedigree,”Theor Appl Genet 95:163-173 (1997). Microsatellites, or SSRs arerelatively short nucleotide sequences, usually from two to three basesin length that are generally repeated in tandem arrays. Amplifiablepolymorphisms are revealed because of differences in the number oftandem repeats that lie between sequences that are otherwise conservedfor each locus. Microsatellite loci are highly polymorphic and areuseful as genetic markers in many plant species, including maize.

The term “mutation” as is used herein refers to a permanent, heritablechange of genetic material, either in a single gene or in the numbers orstructures of the chromosomes.

The term “NILs” as is used herein means near isogenic lines, which arelines of a plant, such as sweet corn, that are genetically identical,except for one locus or a few loci.

The term “nucleotide” as is used herein means the basic building block(subunits) of nucleic acids, such as DNA and RNA. It is an organiccompound that is generally made up of nitrogenous base, a sugar and aphosphate group. DNA molecule consists of nucleotides in which the sugarcomponent is deoxyribose, whereas the RNA molecule has nucleotides inwhich the sugar is ribose. The most common nucleotides are divided intopurines and pyrimidines based upon the structure of the nitrogenousbase. In DNA, the purine bases include adenine and guanine, while thepyrimidine bases are thymine and cytosine. RNA includes adenine,guanine, cytosine and uracil instead of thymine. Aside from serving asprecursors of nucleic acids, nucleotides also serve as importantcofactors in cellular signaling and metabolism. These cofactors includeflavin adenine dinucleotide (FAD), flavin mononucleotide, adenosinetriphosphate (ATP) and nicotinamide adenine dinucleotide phosphate(NADP). To form a DNA or RNA molecule, generally thousands ofnucleotides are joined together in a long chain. A DNA oligonucleotideis a short piece of DNA composed of relatively few (oligo-) nucleotidebases.

The phrase “null hypothesis” as is used herein means a hypothesis whicha researcher tries to disprove, reject or nullify.

The abbreviation “PCR” as is used herein means “polymerase chainreaction,” which is a technique used in molecular biology to amplify asingle copy, or a few copies, of a piece of DNA (a focused segment ofDNA) across several orders of magnitude, generating thousands tomillions of copies of a particular DNA sequence. It is a well-knowntechnique by those having ordinary skill in the art for replicating aspecific piece of DNA in vitro, even in the presence of excessnon-specific DNA. Primers are added (which initiate the copying of eachstrand) along with nucleotides and heat stable Taq polymerase. Bycycling the temperature, the target DNA is repetitively denatured andcopied. Because the newly synthesized DNA strands can subsequently serveas additional templates for the same primer sequences, successive roundsof primer annealing, strand elongation, and dissociation produce rapidand highly specific amplification of the desired sequence. PCR also canbe used to detect the existence of the defined sequence in a DNA sample.A single copy of the target DNA, even if mixed in with other undesirableDNA, can be amplified to obtain billions of replicates. PCR can be usedto amplify RNA sequences if they are first converted to DNA via reversetranscriptase. PCR buffers, primers, probes, controls, markers,amplification kits, sDNA synthesis kits, general PCR kits, and the likeare available from sources that are known by those having ordinary skillin the art, such as Applied Biosystems (Foster City, Calif.), and mayreadily be used by those having ordinary skill in the art in accordancewith the present invention.

The phrase “PCR primer” as is used herein means a short segment of DNAor RNA that is complementary, and hydrogen bonded, to a given DNAsequence, and that is needed to initiate replication by DNA polymerase.It is used to start the PCR process, and acts as a point at whichreplication can proceed.

The term “pest” as is used herein means an animal or insect that isannoying, troublesome, detrimental, harmful, a nuisance, attacks, and/orcauses damage or destruction, for example, to plant crops or livestock.

The term “pericarp” as is used herein means the wall of a plant fruit,such as a corn kernel, which generally is developed from an ovary wall,and contains an outer exocarp, a central mesocarp and an inner endocarp.

The term “phenotype” as is used herein means an observablecharacteristic or trait of an organism, such as sweet corn, such as itsmorphology, development and/or biochemical or physiological properties.It is a biological trait or characteristic possessed by an organism(including a plant) that results from the expression of a specific gene.Phenotypes generally result from the expression of an organism's genes,as well as the influence of environmental factors, and possibleinteractions between the two. In natural populations, most phenotypicvariation is continuous, and is effected by alleles at one or multiplegene loci.

The term “pollen” as is used herein means the fine powder-like materialconsisting of pollen grains that contain the male reproductive cells ofmost plants. Pollen is generally produced by the anthers of seed plants.

The term “pollination” as is used herein means the process by whichplant pollen is transferred, generally from the anther to the stigma(from male reproductive organs to the female reproductive organs) of aplant flower to produce offspring (to form seeds). In flowering plants,pollen is transferred from the anther to the stigma, often by the windor by insects. In cone-bearing plants, male cones release pollen that isusually borne by the wind to the ovules of female cones. The pollengrain generally contains two cells: a generative cell and a tube cell.The generative nucleus generally divides to form two sperm nuclei. Thetube cell generally grows down into the pistil until it reaches one ofthe ovules contained in the ovary. The two sperm generally travel downthe tube and enter the ovule, where one sperm nucleus generally uniteswith the egg. The other sperm nucleus generally combines with the polarnuclei that exist in the ovule, completing a process known as “doublefertilization.” These fertilized nuclei then generally develop into theendocarp, the tissue that feeds the embryo. The ovule itself generallydevelops into a seed that is contained in the flower's ovary (whichripens into a fruit). In gymnosperms, the ovule is exposed (notcontained in an ovary), and the pollen produced by the male reproductivestructures lands directly on the ovule in the female reproductivestructures.

The term “polymorphism” as is used herein means an ability to exist inmore than one form, such as several different forms, and particularlytwo or more clearly different phenotypes existing in the same populationof a species.

The term “polynucleotide” as is used herein means an organic polymermolecule that is composed of nucleotide monomers covalently bonded in achain. DNA and RNA are examples of polynucleotides that have a distinctbiological function.

The term “primer” as is used herein means a relatively shortpre-existing polynucleotide chain to which new deoxyribonucleotides canbe added by DNA polymerase.

The phrases “probes” and “hybridization probes” as are used herein meana fragment of DNA or RNA of a variable length (usually 100-1000 baseslong) which can be radioactively labeled, and can then be used in DNA orRNA samples to detect the presence of nucleotide sequences (the DNAtarget) that are complementary to the sequence in the probe. The probethereby hybridizes to single-stranded nucleic acid (DNA or RNA) whosebase sequence allows probe—target base pairing due to complementaritybetween the probe and target. The labeled probe is first denatured (byheating or under alkaline conditions such as exposure to sodiumhydroxide) into single stranded DNA (ssDNA) and then hybridized to thetarget ssDNA (Southern blotting) or RNA (Northern blotting) immobilizedon a membrane or in situ.

The term “protect” as is used herein means to keep or prevent something(or someone) from causing injury, harm, damage or other adverseconsequences to, for example, an organism, plant, animal or human being.

The term “protein” as is used herein refers to a large molecule composedof one or more chains of amino acids in a specific order, which isdetermined by the base sequence of nucleotides in the gene that iscoding for the protein. Proteins are required for the structure,function, and regulation of cells, and each protein has uniquefunctions.

The term “provide” as is used herein means to supply, or make available,a characteristic, a quality, a thing or the like, that is wanted orneeded, and which previously was not available.

The term “p-value” as is used herein means the probability of obtaininga result equal to or more extreme than what was actually observed, whenthe null hypothesis is true. The p-value is widely used in statisticalhypothesis testing, specifically in null hypothesis significancetesting.

The phrase “quantitative trait locus” (QTL) as used herein means asection of DNA (the locus) that correlates with variations in aphenotype. QTLs are mapped by identifying which molecular markers (suchas SNPs) correlate with an observed trait.

The term “recessive” as is used herein in connection with a gene means agene whose phenotypic effect is expressed in the homozygous state, butis masked in the presence of the dominant allele (i.e. when the organismis heterozygous for that gene). It is a phenotype that is expressed inorganisms (including plants) only if it is homozygous for thecorresponding allele. Usually the dominant gene produces a functionalproduct whereas the recessive allele does not: both one dose and twodoses per nucleus of the dominant allele, therefore, generally lead toan expression of its phenotype, whereas the recessive allele isgenerally observed only in the complete absence of the dominant allele.

The phrase “recurrent parent” as is used herein means the parent towhich a hybrid is crossed in a backcross. If the recurrent parent has anelite genotype, at the end of a backcrossing program, an elite genotypeis typically recovered.

The term “restriction enzyme” as is used herein means a protein (enzyme)produced by bacteria that cuts DNA at or near specific recognitionnucleotide sequences known as restriction sites (i.e., it acts likescissors).

The term “seed” as is used herein means a propagating organ formed inthe sexual reproductive cycle of gymnosperms and angiosperms (male andfemale sex cells) that includes a protective coat enclosing an embryoand food reserves. It is a small hard fruit that is generally located ina fertilized ovule of a plant. A seed has two main components, theembryo and the endosperm. The endosperm acts as a food store for theembryo which, over time, will grow from this rich food supply thatenables it to do so. The seed contains an embryo and, in most plants,stored food reserves wrapped in a seed coat. Under favorable growthconditions, a seed begins to germinate, and the embryonic tissues resumegrowth, developing towards a seedling.

The term “selection” as is used herein means the preferential survivaland reproduction, or preferential elimination, of individuals withcertain genotypes (genetic compositions) by means of natural orartificial controlling factors.

The term “selfing” as is used herein means manually pollinating a plantby placing its pollen on its own stigma.

The term “self-pollination” is as used herein refers to the transfer ofpollen from the anther to the stigma of the same flower, another floweron the same plant, or the flower of a genetically identical plant.

The term “silk” as is used herein in regard to maize means the longfilamentous styles and stigmas that appear as a silky tuft or tassel atthe tip of an ear of corn.

The abbreviation “SNP” as is used herein means “single nucleotidepolymorphism,” and is a DNA sequence variation occurring when a singlenucleotide (A, T, C, or G) in the genome (or other shared sequence)differs between members of a species (or between paired chromosomes inan individual). For example, at a specific base position in the humangenome, the base C may appear in most individuals, but in a minority ofindividuals, the position is occupied by base A. There is a SNP at thisspecific base position, and the two possible nucleotide variations (C orA) are said to be alleles for this base position.

The phrase “test cross” as is used herein means the crossing of anorganism, such as a plant, with an unknown genotype, to a homozygousrecessive organism (tester). It is a cross between an individual ofunknown genotype or a heterozygote (or a multiple heterozygote) to ahomozygous recessive individual.

The term “transcription” as is used herein means the synthesis of RNAunder the direction of DNA. RNA synthesis, or transcription, is theprocess of transcribing DNA nucleotide sequence information into RNAsequence information. Both nucleic acid sequences use complementarylanguage, and the information is simply transcribed, or copied, from onemolecule to the other. DNA sequence is enzymatically copied by RNApolymerase to produce a complementary nucleotide RNA strand (messengerRNA or mRNA) because it carries a genetic message from the DNA to theprotein-synthesizing machinery of the cell.

The term “translation” as is used herein means the process by whichpolypeptide chains are synthesized, the sequence of amino acids beingdetermined by the sequence of bases in a messenger RNA, which in turn isdetermined by the sequence of bases in the DNA of the gene from which itwas transcribed.

The term “whorl” as is used herein means a configuration of a maizeplant just prior to an extrusion of its tassel (i.e., when the leavesare very concentrated, and there are multiple layers of them, and thetassel will soon be visible, and the internotes of the plant willelongate).

The term “wholesome” as is used herein in regard to maize means notunhealthy for the human or animal body, or healthy for the human oranimal body (i.e., contributing to, enhancing, promoting or providingphysical, mental and/or emotional well-being).

The term “wild-type” as is used herein refers to a native or predominantgenetic constitution before mutations, usually referring to the geneticconstitution normally existing in nature.

The term “yield” as is used herein refers to plant, plant materialand/or seed productivity, such as the productivity per unit area of aparticular plant product of commercial significance.

Description of the Invention

The following description illustrates the methods and products of thepresent invention. The description is intended to be merely illustrativeof the present invention, and not limiting in either scope or spirit.Those of ordinary skill in the art will readily understand thatvariations of certain of the conditions and/or steps employed in theprocedures described herein can be employed. While these procedures havebeen performed using sweet corn kernels and plants, the same proceduresthat are described may be employed with other members of the maizefamily, including field, flint, flour, dent, pop, waxy and pod corn.

The present invention may be understood more readily by reference to thefollowing detailed description of the steps undertaken to provide ameans for controlling and defending against the corn silk fly and phoridfly in maize plant lines, and means of testing and identifying thepresence of the genes responsible for controlling and defending againstthe corn silk fly and phorid fly. The following detailed descriptionalso describes the products of the invention that incorporate the genesresponsible for those control and defense mechanisms.

Procurement of Maize Parent Inbred Lines for Crossing and InitialSelections

Maize inbred lines designated IN 705, IN 706 and IN 714 were obtainedfrom Dr. W. Paul Williams, Supervisory Research Geneticist USDA-ARS,Corn Host Plant Resistance Unit (CHPRU) (Mississippi State, MS). Theseinbred lines are all dent corn inbred lines and are publicly availablefrom the CHPRU at Mississippi State, MS. Sixteen maize inbred lines fromAbbott & Cobb's (Feasterville, Pa.) sweet corn breeding program,including AC 194, BL 6440 (181MR) and AC 232Y, were then selected forbackcrossing into these dent corn lines.

Breeding Protocol for Maize Inbred Parent Lines

The maize inbred parent lines identified in TABLE 1 below werebackcrossed and self-pollinated at the Everglades Research and EducationCenter, IFAS, University of Florida (Belle Glade, Fla.) or at the Abbott& Cobb, Inc. (A&C) Research Breeding Station (Loxahatchee, Fla.), in themanner described in TABLE 2. Because dent corn possesses many traitsthat are not suitable for sweet corn (which must be sweet and mustmaintain its sweetness and delay or reduce conversion of sugars tostarches in order to be palatable and acceptable to consumers), theselection process required a focus not only on identifying insectprotection efficacy but rigorous selection for the necessary sweet cornhorticultural attributes to make the corn acceptable to consumers.Utilizing these selection criteria, the progeny of AC 194, BL 6440(181MR) and AC 232Y (out of the sixteen maize inbred lines initiallychosen from A&C's sweet corn breeding program) were ultimately selectedfor further inbred line development.

TABLE 2 shows the maize breeding protocol for backcrossing and selectionof the parent inbred lines obtained from the sources described in TABLE1.

TABLE 1 Donor Maize Parent Inbred Lines Designation Source Type IN 705CHPRU Dent IN 706 CHPRU Dent IN 714 CHPRU Dent AC 194 A&C Sweet BL 6440(181MR) A&C Sweet AC 232Y A&C Sweet

TABLE 2 Breeding Protocol Step Activity Result 1 A&C commercial sweetcorn parent inbred F1 Hybrid line X dent corn parent inbred line(TABLE 1) 2 F1 - Backcrossed to recurrent parent (A&C BC1 (Back-Cross 1)commercial parent inbred line) 3 Self-pollination of BC1 S1(Self-Pollination 1) 4 Field insect protection efficacy testing viaUnsuccessful Inbred natural corn silk fly infestation including LinesEliminated selection for horticultural attributes 5 Continuedbackcrossing to the recurrent BC2-BC6 and parent and self-pollinationcombined with S2-S6 field insect protection efficacy testing withnatural corn silk fly and phorid fly infestation including selection forsweet corn horticultural attributes 6 Repetition of breeding listedabove until six Successful Inbred backcross generations were completedand Lines reconstitution of the recurrent parents was consideredgenetically stable, uniform and complete

Plantings of Parent Inbred Lines and Developed Hybrid Lines in TestNurseries

The initial crosses of the dent corn inbred parent lines and the sixteensweet corn inbred lines, the backcrossing of the initial F1 hybrids withthe recurrent sweet corn inbred lines, and all further backcrossing andrecurrent selections described in steps 1, 2, 4, 5 and 6 of TABLE 2,were performed in the test nurseries at Belle Glade or Loxahatchee, Fla.The nurseries consisted of fifty single rows, approximately 25 feetlong, each containing about 30 plants. The plant spacing within the rowswas approximately 8 to 8 1/2″, and the spacing between (or width of) therows was about 30″. The plantings were staged so that pollinationoccurred during the times of the season in Florida when corn silk fliesand phorid flies were most prevalent (i.e., “natural” infestation wasemployed). Damage assessments were made from the time the plants were inthe whorl state until the time of the final ear harvest. No chemicalpesticides and/or insecticides were applied aerially, on the ground, orotherwise to any of the maize plants, and/or to any other surroundingsto control the corn silk fly and phorid fly populations at eitherlocation.

Damage Rating Scale and Phenotypic Analyses of Resulting Maize PlantEars and Kernels

A damage rating scale was developed to evaluate the extent of damage tothe ears and kernels of a maize plant attributable to insect damage tothe maize plant ears and ear tips and maize kernels, and to assist inmaking selections during the recurrent selection breeding process.

The damage rating scale was a survey-type phenotypic scale that includednine discrete categories, or injury classes, ranging from “0” to “8,”with a focus on the maize plant ear tips. The damage rating scalemeasured the depth of insect feeding from the tip of the ear downwardstowards the stalk. The maize plant ears and kernels were rated at thematurity stage of the ear.

The damage rating scale was developed within the framework andassumptions of Poisson Distribution. Poisson Distribution is well knownto those of ordinary skill in the art. (See R. G. D. Steel et al.,“Principles and Procedures of Statistics: A Biometrical Approach,”

McGraw-Hill Publ. Co., New York, 2nd ed., ISBN 0-070-60926-8, 629(1980)); and G. W. Snedecor et al., “Statistical Methods,” Iowa StateUniv. Press, Ames, 7th ed., ISBN 0-813-81560-6, 505 (1980).) Toestablish the damage rating scale, precise metrics and numerical valueswere assigned to the insect damage that was visually observed to boththe ears and kernels of the maize plants.

TABLE 3 below shows the damage rating scale developed, and utilized, toevaluate and rate the maize plants described in Steps 1, 2, 4, 5 and 6of TABLE 2 in terms of insect damage and to assist in the selection ofmaize parent lines and maize hybrids. Ears that received a rating of “0”or “1” are considered to be commercially acceptable to consumers andseed from those ears was selected for use in further breeding efforts toproduce maize parental inbred lines. Ears that received ratings of 2-8are considered to have too much insect damage to be commerciallyacceptable to consumers and were therefore not selected to be used infurther breeding efforts.

TABLE 3 Ear and Kernel Phenotypic Insect Damage Rating Scale (25Ears/Row Average) Maize Ear/Kernel Damage Rating Scale No Damage To AnyEars 0 Less than 30 mm Damage to Ear Tips of 1-3 1 Ears Less than 30 mmDamage to Ear Tips of 4-6 2 Ears Ear Tip Damage to 4-7 Ears Extendingfrom 3 the First to Third Kernel Ear Tip Damage to 4-7 Ears Extendingfrom 4 the First to Fourth Kernel Ear Tip Damage to 7-10 Ears Extendingfrom 5 the First to Fourth Kernel Ear Tip Damage to 7-10 Ears Extendingfrom 6 the First to Sixth Kernel Ear Tip Damage to All Ears With theMajority 7 of Ears Having Kernel Damage Beyond the Fourth to SixthKernel Ear Tip Damage to All Ears with all Ears 8 Having Kernel DamageBeyond the Fourth to Sixth KernelThe above damage rating scale is independent of any standards for freshproduce used by the

Agricultural Marketing Service of the United States Department ofAgriculture (USDA).

Genotypic Analyses of Resulting Leaf Tissue Samples

Based on the damage rating scale in TABLE 3, numerous selections weremade to identify the maize plants within each row that achieved thehighest level of insect protection. Leaf tissue samples were only takenfrom the best (highest rated) (rated as a “0” and “1” in TABLE 3) of theabove lines, as well as those that were the lowest rated (for a relativecomparison) (those rated as a “7” or “8” in TABLE 3). Approximately10-15 leaf tissue samples from each row were provided to RAPiD Genomics(Gainesville, Fla.), which was contracted to provide commercial genomesequencing services. Initially, 881 leaf tissue samples were provided toRAPiD Genomics for purposes of performing a GWAS to identify the genesassociated with the control of and defense against insects that had beenobserved in testing plots. From these 881 leaf tissue samples, thesamples were reduced to 784 tissue samples using phenotypic informationregarding desired horticultural characteristics.

RAPiD Genomics utilized its genotyping method called “Capture-Seq” toidentify the DNA markers having significant association with the controlof and defense against insect damage across all ten maize chromosomespresent in the 784 leaf tissue samples. Using methods known to those ofordinary skill in the art, DNA was extracted from each of the 784 leaftissue samples. The extracted DNA was then fragmented using knownmethods to an average size of 300 bp using ligases (enzymes).Next-Generation Sequencing libraries were then prepared.

A total of 837,779 probes were designed in 39,024 protein-coding genesannotated in the well-known B73 maize cultivar. These probes werefiltered to obtain high-quality baits, resulting in 60,433 probes. Outof the 60,433 probes, 5,000 were randomly chosen limiting the selectionto one probe per gene. In addition, a set of probes was designed totarget peptidase and inhibitor genes in maize. After filtering, 617additional high quality probes were designed, resulting in 5,617 probesfor use in hybridizing target areas of interest in the 784 tissuesamples.

The Next-Generation Sequencing libraries prepared from the DNA extractedfrom the 784 leaf tissue samples were then hybridized against the 5,617probes, and the enriched product was sequenced on an Illumina platform(2×100 bp mode). The resulting raw DNA sequencing data were processed ina bioinformatics pipeline using common techniques—read qualityfiltering, data alignment against the B73 reference genome, and Bayesianvariant culling at the population level, resulting in the identificationof a total of 46,069 SNPs. The resulting SNP data was then adjusted inthe GWAS analysis for association with the phenotypic informationderived from the damage rating scale data.

The phenotypic data generated from the insect damage rating scale (TABLE3) was used to create two scoring scales to be used in the GWASanalysis. The first insect damage scoring scale ranged from 1-8 (Phe1-8) (“the first scoring scale”), in which a score of 1 indicated amaize plant that had the highest level of insect defense and a score of8 indicated a maize plant that was the most susceptible (least protectedor had the least amount of defense) to damage.

In addition, a second scoring scale was established to create a binarytrait for purposes of the GWAS analysis (“the second scoring scale”)by: 1) recoding and grouping all plants having scores of 0 or +1 underthe first scoring scale together (i.e., by giving a score of “0” tothose maize plants that had a rating of either 0 or 1 in the firstscoring scale); and 2) recoding and grouping together every maize planthaving a rating score greater than 1 on the first scoring scale a scoreof “1” (PheBIN).

Both phenotype data sets were then analyzed independently as dependentvariables of a mixed linear regression model, testing the associationlevel of each SNP with the phenotype (TABLE 3). A relationship matrixbuilt from the markers was also adjusted in the model to correct forpopulation structure. The p-values for each marker were calculated toinfer the probability of association of that marker with the givenattribute (highest defense to damage).

All of the SNPs were adjusted in a GWAS analysis to test for anassociation with both phenotypic data sets from the two phenotypescoring scales (Phe1-8 and PheBIN). The resulting data were then used toidentify candidate QTL regions.

GWAS Analyses

FIG. 1 is a representation of a statistical model of a GWAS showing anassociation of each of the above SNPs across all ten chromosomes inmaize plants. This GWAS analysis was conducted with the data setdiscussed above in connection with the first scoring scale (Phe1-8). InFIG. 1, the x-axis represents the location of each SNP across the tenmaize plant chromosomes relative to the SNP's association with insectdamage defense based upon its phenotypic scoring. The y-axis illustratesthe relative probability (-log of the p-values) of each such SNP.

FIG. 2 is a representation of a statistical model of a GWAS showing anassociation of each of the above SNPs across all ten chromosomes presentin maize plants. This GWAS analysis was conducted with the binary dataset discussed above in connection with the second scoring scale(PheBIN). This GWAS analysis revealed one additional candidate QTLregion.

Based on the GWAS analyses, seven SNPs having a highly positiveassociation (associated with a p-value less than 1×10⁻⁶) with the maizeplant insect defense phenotype were identified. These seven SNPs arelocated on four maize chromosomes (chromosomes 3, 4, 7, and 9). QTLregions on two of these four chromosomes (chromosomes 7 and 9) werepreviously reported to be candidate regions for the resistance to bothfall armyworm and southwestern corn borer. (Brooks et al. 2007). Theidentification of these same QTL regions on chromosomes 7 and 9 in theGWAS analysis substantiates the significant association between theseregions of the genome and the expression of a means of controlling anddefending against insect damage.

The list of SNPs corresponding to QTL regions with significantassociations to maize plant insect defense phenotypes and theircorresponding genes are presented on TABLE 4.

TABLE 4 SNPs having a Highly Positive Association tothe Maize Plant Insect Defense Phenotype Maize Plant Chromosome p-Link the SNP Alternative Number value B73_Bin gene ID DesignationAlleles 3 3.04E-07 3.04 SEQ ID NO.: 1  17606436 C SEQ ID NO.: 6 32.96E-06 3.04 SEQ ID NO.: 1  17606457 TGCG SEQ ID NO.: 7 4 8.13E-06 4.01SEQ ID NO.: 2   4808588 C SEQ ID NO.: 8 4 7.14E-07 4.01 SEQ ID NO.: 2  4808626 A SEQ ID NO.: 9 7 1.98E-06 7.02 SEQ ID NO.: 3 126746831 GSEQ ID NO.: 10 7 9.08E-06 7.03 SEQ ID NO.: 4 143394449 ATASEQ ID NO.: 11 9 6.76E-06 9.02 SEQ ID NO.: 5  19033476 TTGCAGTSEQ ID NO.: 12

Production of Maize Hybrids Providing Defense to Corn Silk Fly andPhorid Fly

After identifying the SNPs having a highly positive association withcorn silk fly, phorid fly and other insect defense, the inbred maizeplant lines containing the genes for controlling and defending againstthe corn silk fly and phorid fly were identified and selected for hybridproduction. Maize inbred parent lines, 1115, 1118 and 1150, wereidentified as containing the corn silk fly and phorid fly defense genes.These inbred parent lines have been designated NBD 01, NBD 02 and NBD03, respectively, for further breeding and hybrid production.

Maize Inbred Parent Line 1115 (NBD 01)

Maize inbred parent line 1115 (NBD 01) resulted from an initial cross ofBL 6440 (181MR) with IN705-2 (BL 6440 (181MR) X IN705-2). The initialcross was made at Belle Glade, Fla. in the Spring of 2014. Backcrossesto the recurrent parent (BL 6440 (181MR)) were made in the Fall of 2014,the Spring of 2015, the Summer of 2015 in Illinois, and the Spring of2016 in Belle Glade.

Maize Inbred Parent Line 1118 (NBD 02)

Maize inbred parent line 1118 (NBD 02) resulted from an initial cross ofAC 194 with

IN706-A2 (AC 194 X IN706-A2). The initial cross was made at Belle Glade,Fla. in the Spring of 2014. Backcrosses to the recurrent parent (AC 194)were made in the Fall of 2014, the Spring of 2015, the Summer of 2015 inIllinois, and the Spring of 2016 in Belle Glade.

Maize Inbred Parent Line 1150 (NBD 03)

Maize inbred parent line 1150 (NBD 03) resulted from an initial cross ofAC 232Y with

IN714-1 (AC 232Y X IN714-1). The initial cross was made at Belle Glade,Fla. in the Spring of 2014. Backcrosses to the recurrent parent (AC232Y) were made in the Fall of 2014, the Spring of 2015, the Summer of2015 in Illinois, and the Spring of 2016 in Belle Glade.

Maize Hybrids (NBDX 1001 and NBDX 1002)

Two hybrids were produced in the Spring of 2016 at the Belle Glade, Fla.nursery using 1115, 1118 and 1150 as maize inbred parent lines. Thehybrid designated NBDX 1001 was produced using 1115 (NBD 01) as thefemale parent and 1118 (NBD 02) as the male parent. The hybriddesignated NBDX 1002 was produced using 1118 (NBD 02) as the femaleparent and 1150 (NBD 03) as the male parent. The most desirable hybridsproduced had both: (i) the highest level of corn silk fly and phorid flydefense (as evidenced by their scores on the first scoring scale); and(ii) the most favorable, commercially-required horticulturalcharacteristics.

Maize Hybrids (NBX 2001 and NBX 2002)

In addition to maize hybrids having two parent inbred lines each ofwhich contain the genes for controlling and defending against the cornsilk fly and phorid fly, maize hybrids were produced using one parentinbred line containing the genes for controlling and defending againstthe corn silk fly and phorid fly and existing commercial inbred maizelines.

NBX 2001

Inbred parent line 1115 (NBD 01) was crossed with existing commercialinbred line, AC 266Y. The resulting single cross maize hybrid createdusing the 1115 (NBD 01) inbred line as the male parent, and AC 266Y asthe female parent, designated as NBX 2001, had a score on the firstscoring scale of either 0 or 1.

NBX 2002

Inbred parent line 1118 (NBD 02) was crossed with existing commercialmaize line, AC 274Y ST. The resulting single cross maize hybridvarieties created using the 1118 (NBD 02) inbred line as the maleparent, designated as NBX 2002, and AC 274Y ST as the female parent,designated as NBX 2002, had a score on the first scoring scale of either0 or 1.

The specific pedigrees and the rows where the progeny were located areset forth in TABLE 5.

TABLE 5 Corn Silk Fly Defensive Hybrids Produced Parent Inbred SourceRows in Hybrid Designation Designations Nursery NBDX 1001 NBD 01 × NBD02 16FF 1115 × 1118 NBDX 1002 NBD 02 × NBD 03 16FF 1118 × 1150 NBX 2001AC 266Y × NBD 01 16FF 1045 × 1115 NBX 2002 AC 274Y ST × NBD 02 16FF 1057× 1118

Phenotypic Comparison with Commercially Available GMO Maize Varieties

To determine how maize varieties containing the identified SNPs compareto commercially available GMO hybrids containing insect protectiontraits, a comparative trial was conducted. Commercially available maizeGMO hybrids from Syngenta (Boise, Id.) (GSS-0966) and Seminis (St.Louis, Mo.) (SV9010) were planted at both the Belle Glade andLoxahatchee, FL testing locations to make phenotypic comparisons ofinsect damage to the maize hybrids described above under the same insectpressure and conditions. Phenotypic information was obtained for themaize plants in each of the breeding plots. The sweet corn which was theproduct of the recurrent selection process described in TABLE 2 scoredlower on the insect damage rating scale than the GMO maize hybrids grownunder the same testing environment.

Preferred Embodiment

The preferred embodiment of the invention is one where all seven SNPsare identified as providing a means of controlling and defending againstthe corn silk fly and phorid fly. However, each of the seven SNPsconfers some means of controlling and defending against the corn silkfly and phorid fly. Therefore, the invention is not limited to thepreferred embodiment but extends to methods and products for controllingand defending against the corn silk fly and phorid fly, whichincorporate one or more of the seven SNPs identified herein.

Use of SNPs as Molecular Markers for Maize Hybrid Development

Having identified the SNPs corresponding to QTL regions with significantassociations to maize plant insect defense phenotypes and theircorresponding genes, these SNPs can be used in marker assisted breedingand introgression strategies to reduce the time for and increase theefficiency of producing new maize parental inbred lines and maizehybrids having the means for controlling and defending against the cornsilk fly and phorid fly. The use of molecular markers for the purpose ofmore efficiently producing parental plant lines is well known anddescribed in the art. Identification of the relevant SNPs is frequentlythe limiting factor in using molecular markers that are responsible forthe desired characteristic to be introduced. The present inventionidentifies the relevant SNPs that provide a means of controlling anddefending against the corn silk fly and phorid fly, which were not knownprior to the invention.

Identification of the seven SNPs having a high degree of associationwith control of and defense against the corn silk fly and phorid flyprovides one of ordinary skill in the art the means for testing andidentifying the presence of the genes responsible for controlling anddefending against the corn silk fly and phorid fly in maize plants. Theability to test for and identify these genes is highly useful andadvantageous to plant breeders seeking to develop maize plant lines thatcontrol for and defend against the corn silk fly and phorid fly.

Sources of Materials and Equipment

All of the materials and equipment that are employed in the methods andused for producing the products of the invention are commerciallyavailable from sources known to those having ordinary skill in the art.The initial seed sources were from the Corn Host Plant Resistance Unitat Mississippi State, MS and from A&C's commercial sweet corn breedingprogram. RAPiD Genomics's “Capture-Seq” genotyping method was utilizedto identify the SNPs. Capture-Seq is a commercial service provided byRAPiD Genomics.

Seed Deposits of Parental Inbred Lines and Initial Hybrids

Because seed is living material, and must be produced over time, thereare only limited quantities of the inbred lines 1115, 1118, and 1150available for deposit at the present time. There are also limitedquantities of maize hybrids containing one or more of the foregoingparental inbred lines available for deposit. For these reasons, seeddeposits have been made with the American Type Culture Collection (ATCC)in the following amounts: ATCC Patent Deposit Designations PTA-124546(AC 266Y) (2,000 seeds), PTA-124547 (AC 274YST) (2,000 seeds),PTA-124548 (NBD 01) (2,000 seeds), PTA-124549 (NBD 02) (2,000 seeds) andPTA-124550 (NBD 03) (200 seeds). The amounts deposited will besupplemented after seed increases are available in the future.

While the present invention has been described herein with specificity,and with reference to certain preferred embodiments thereof, those ofordinary skill in the art will recognize numerous variations,modifications and substitutions of that which has been described whichcan be made, and which are within the scope and spirit of the invention.It is intended that all of these modifications and variations be withinthe scope of the present invention as it is described and claimedherein, and that the invention be limited only by the scope of theclaims which follow, and that such claims be interpreted as broadly asis reasonable.

What is claimed is:
 1. A hybrid maize plant, plant material or seedcomprising quantitative trait locus regions that control or defendagainst corn silk fly and phorid fly.
 2. A male or female inbred maizeparent line comprising quantitative trait locus regions that control ordefend against corn silk fly and phorid fly.
 3. A hybrid maize plant,plant material or seed of claim 1 wherein the maize plant is classifiedas Zea mays.
 4. A hybrid maize plant, plant material or seed of claim 1wherein the maize plant is classified as Zea mays, convar saccharatavar. rugosa.
 5. A hybrid maize plant, plant material or seed of claim 1wherein the maize seed is Sweet Corn Hybrid NBDX
 1001. 6. A hybrid maizeplant, plant material or seed of claim 1 wherein the maize seed is SweetCorn Hybrid NBDX
 1002. 7. A male or female inbred maize parent line ofclaim 2 wherein the maize plant is classified as Zea mays.
 8. A male orfemale inbred maize parent line of claim 2 wherein the maize plant isclassified as Zea mays, convar saccharata var. rugosa.
 9. A male orfemale inbred maize parent line of claim 2 wherein the maize seed isSweet Corn NBD
 01. 10. A male or female inbred maize parent line ofclaim 2 wherein the maize seed is Sweet Corn NBD
 02. 11. A male orfemale inbred maize parent line of claim 2 wherein the maize seed isSweet Corn NBD
 03. 12. A hybrid maize plant, plant material or seed ofclaim 1, wherein the ear and kernel have a damage rating of 2 or less.13. A hybrid maize plant, plant material or seed of claim 1, wherein theear and kernel have a damage rating of 1 or less.
 14. A male or femaleinbred maize parent line of claim 2, wherein the ear and kernel have adamage rating of 2 or less.
 15. A male or female inbred maize parentline of claim 2, wherein the ear and kernel have a damage rating of 1 orless.
 16. A hybrid maize plant, plant material or seed of claim 1,wherein the genetic background comprises SNPs or indels including one ormore of SEQ ID NOS. 6-12.
 17. A method for producing a hybrid maizeplant, plant material or seed comprising quantitative trait locusregions that control or defend against corn silk fly and phorid fly,comprising the following steps: (a) identifying one or more maize parentlines containing one or more of the SNPs or indels that have beenidentified as providing control or defense against corn silk fly andphorid fly utilizing molecular markers; (b) incorporating one or more ofthe SNPs or indels that provide control or defense against corn silk flyand phorid fly into a male or female inbred maize parent line; (c)crossing the male or female inbred maize parent line containing one ormore of the SNPs or indels that provide control or defense against cornsilk fly and phorid fly with another inbred maize parent line to producea hybrid; and (d) optionally, conducting one or more geneticidentification tests to confirm that the hybrid contains the SNPs orindels that provide control or defense against corn silk fly and phoridfly.
 18. The method of claim 17, wherein the hybrid maize plant, plantmaterial or seed is Zea mays.
 19. The method of claim 17, wherein thehybrid maize plant, plant material or seed is Zea mays, convarsaccharata var. rugosa.
 20. A method for producing a male or femaleinbred maize parent line that provides control or defense against cornsilk fly and phorid fly, comprising the following steps: (a) identifyingone or more maize lines containing one or more of the SNPs or indelsthat have been identified as providing control or defense against cornsilk fly and phorid fly utilizing molecular markers; (b) incorporatingone or more of the SNPs or indels that provide control or defenseagainst corn silk fly and phorid fly into a male or female inbred maizeparent line; (c) utilizing recurrent selection or other plant breedingmethods to produce a male or female inbred maize parent line containingone or more of the SNPs or indels that provide control or defenseagainst corn silk fly and phorid fly; and (d) optionally, conducting oneor more genetic identification tests to confirm that the inbred maizeparent line contains the SNPs or indels that provides control or defenseagainst corn silk fly and phorid fly.
 21. The method of claim 20,wherein the inbred maize plant, plant material or seed is Zea mays. 22.The method of claim 20, wherein the inbred maize plant, plant materialor seed is Zea mays, convar saccharata var. rugosa.
 23. A method ofclaim 17, wherein the hybrid maize plant, plant material or seed iscapable of producing an ear and kernel having a damage rating of 2 orless.
 24. A method of claim 17, wherein the hybrid maize plant, plantmaterial or seed is capable of producing an ear and kernel having adamage rating of 1 or less.
 25. A method of claim 20, wherein the maleor female inbred maize parent line is capable of producing an ear andkernel having a damage rating of 2 or less.
 26. A method of claim 20,wherein the male or female inbred maize parent line is capable ofproducing an ear and kernel having a damage rating of 1 or less.
 27. Ahybrid maize plant, plant material or seed prepared by a processcomprising the steps of: (a) identifying one or more maize parent linescontaining one or more of the SNPs or indels that have been identifiedas providing control or defense against corn silk fly and phorid flyutilizing molecular markers; (b) incorporating one or more of the SNPsor indels that provide control or defense against corn silk fly andphorid fly into a male or female inbred maize parent line; (c) crossingthe male or female inbred maize parent line containing one or more ofthe SNPs or indels that provide control or defense against corn silk flyand phorid fly with another inbred maize parent line to produce ahybrid; and (d) optionally, conducting one or more geneticidentification tests to confirm that the hybrid contains the SNPs orindels that provide control or defense against corn silk fly and phoridfly.
 28. The product of claim 27, wherein the hybrid maize plant, plantmaterial or seed is Zea mays.
 29. The product of claim 27, wherein thehybrid maize plant, plant material or seed is Zea mays, convarsaccharata var. rugosa.
 30. A male or female inbred maize parent lineprepared by a process comprising the steps of: (a) identifying one ormore maize lines containing one or more of the SNPs or indels that havebeen identified as providing control or defense against corn silk flyand phorid fly utilizing molecular markers; (b) incorporating one ormore of the SNPs or indels that provide control or defense against cornsilk fly and phorid fly into a male or female inbred maize parent line;(c) utilizing recurrent selection or other plant breeding methods toproduce a male or female inbred maize parent line containing one or moreof the SNPs or indels that provide control or defense against corn silkfly and phorid fly; and (d) optionally, conducting one or more geneticidentification tests to confirm that the inbred maize parent linecontains the SNPs or indels that provide control or defense against cornsilk fly and phorid fly.
 31. The product of claim 30, wherein the inbredmaize plant, plant material or seed is Zea mays.
 32. The product ofclaim 30, wherein the inbred maize plant, plant material or seed is Zeamays, convar saccharata var. rugosa.
 33. A product of claim 27, whereinthe hybrid maize plant, plant material or seed is capable of producingan ear and kernel having a damage rating of 2 or less.
 34. A product ofclaim 27, wherein the hybrid maize plant, plant material or seed iscapable of producing an ear and kernel having a damage rating of 1 orless.
 35. A product of claim 30, wherein the male or female inbred maizeparent line is capable of producing an ear and kernel having a damagerating of 2 or less.
 36. A product of claim 30, wherein the male orfemale inbred maize parent line is capable of producing an ear andkernel having a damage rating of 1 or less.